An antibacterial peptide-like and application thereof

Abstract

The application belongs to the technical field of anti-infection of antibacterial peptide drugs, and provides an antibacterial peptide and application thereof.The antibacterial peptide takes an olefin functionalized amino acid as a monomer, and is obtained through a free radical polymerization reaction.The antibacterial peptide shows no inhibition on growth of gram-positive bacteria and gram-negative bacteria, but has an effect of clearing bacterial biofilms, and has excellent biocompatibility and no toxicity.The raw materials and reagents of the application are easy to obtain, and the preparation process is simple, so that the application has wide prospects in the fields of clinical infection treatment, environmental protection and food safety.

Description

Technical Field

[0001] This invention relates to the field of antimicrobial peptide drugs for anti-infection technology, and more particularly to an antimicrobial peptide and its application. Background Technology

[0002] Bacterial biofilms, also known as biofilms, are widely present on various moist surfaces containing water, such as food, food processing equipment, water pipes, industrial pipelines, ventilation equipment, medical devices, and even the surfaces of human tissues and organs in pathological states. They are structured bacterial communities composed of bacterial cells attached to the surface of inert or active entities and a hydrated matrix encapsulating the bacteria. Almost all bacteria can form biofilms under certain conditions. Salmonella, Escherichia coli, Listeria, and Staphylococcus aureus are common pathogens that easily cause foodborne illnesses. They exist in the air, water, dust, and human and animal excrement, making food highly susceptible to contamination. For example, food processing workers, cooks, or sales personnel may carry bacteria, causing food contamination; food may be contaminated before processing or during processing, producing toxins and causing food poisoning; improperly packaged cooked food may be contaminated during transportation; and livestock may be contaminated before slaughter. Furthermore, bacteria easily form biofilms on food, various food processing surfaces, and non-food processing surfaces (such as walls, sewers, and dead corners), which can then contaminate food through hands or air, causing food poisoning. The metabolic activities of bacteria in biofilms can not only corrode pipes and metal surfaces but may also lead to diseases in animals, plants, and humans.

[0003] Antimicrobial peptides possess broad-spectrum antimicrobial activity and exhibit strong bactericidal effects, particularly their effectiveness against certain drug-resistant pathogens, which has attracted considerable attention. As a novel antimicrobial agent, the use of antimicrobial peptides offers a new approach and method for treating biofilm infections. Antimicrobial peptides demonstrate unique advantages in inhibiting biofilm formation, killing bacteria within biofilms, and eliminating mature biofilms. However, due to the strong survival ability of bacterial biofilms, the ineffectiveness of conventional antibiotics against biofilms, drug resistance resulting from long-term use, and the harm caused to patients by high doses, existing antimicrobial agents are not ideal in clearing bacterial biofilms. Therefore, developing an antibiofilm agent with excellent clearance efficacy has become an urgent problem for those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides an antimicrobial peptide and its application, the purpose of which is to solve the technical problem that existing antimicrobial agents have poor removal effects on bacterial biofilms.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides an antimicrobial peptide with the function of clearing bacterial biofilms, having the general structural formula shown in formula (I):

[0007]

[0008] Where n is 5 to 100; the structural formula of R is shown in equations (II) to (V):

[0009]

[0010] This invention provides the application of the above-mentioned antimicrobial peptides in clearing bacterial biofilms, wherein the bacteria in the bacterial biofilm include Gram-negative bacteria and / or Gram-positive bacteria.

[0011] Furthermore, the preparation method of the antimicrobial peptide includes the following steps:

[0012] S1. The amino acid, olefin compound and water are mixed and reacted to obtain the reaction product. The reaction product is then sequentially extracted, dried, rotary evaporated and vacuum dried to obtain olefin-functionalized amino acid monomer.

[0013] S2. An antimicrobial peptide is obtained by mixing olefin-functionalized amino acid monomers, chain transfer agents, catalysts and solvents and then carrying out a polymerization reaction.

[0014] Furthermore, in step S1, the amino acid includes N... α -tert-Butyloxycarbonyl-D-lysine, N ε -tert-Butyloxycarbonyl-D-lysine, N α -tert-Butyloxycarbonyl-D-tyrosine, N α -tert-Butyloxycarbonyl-D-2,3-diaminopropionic acid and N β Any one of -tert-butoxycarbonyl-D-2,3-diaminopropionic acid;

[0015] The olefin compound contains acryloyl chloride and / or methacryloyl chloride.

[0016] Furthermore, in step S1, the ratio of amino acids, olefin compounds, and water is 800–1200 mg: 300–600 mg: 3–10 mL.

[0017] Furthermore, in step S1, the reaction temperature is 0–30°C, and the reaction time is 3–8 hours.

[0018] Furthermore, in step S2, the chain transfer agent includes one or more of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid, 2-(dodecylthiocarbonylthiothio)propionic acid, S-(2-cyano-2-propyl)-S-dodecyltrithiocarbonyl ester, and 2,2'-[methylthiobis(thio)]bis[2-methylpropionic acid];

[0019] The catalyst includes one or more of 2′,4′,5′,7′-tetrabromofluorescein, tetraphenylporphyrin zinc, tris(2-phenylpyridine)iridium, sodium bicarbonate, ammonium persulfate, and azobisisobutyronitrile;

[0020] The solvent includes one or more of ethyl acetate, methanol, ethanol, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, concentrated hydrochloric acid, and hydrogen peroxide.

[0021] Furthermore, in step S2, the molar ratio of the olefin-functionalized amino acid monomer to the chain transfer agent is 20-150:1;

[0022] The molar ratio of the olefin-functionalized amino acid monomer to the catalyst is 10. 3 ~10 5 :1;

[0023] The mass-to-volume ratio of the olefin-functionalized amino acid monomer to the solvent is 0.5–1.5 g: 7 mL.

[0024] Furthermore, in step S2, the polymerization reaction takes 4 to 12 hours.

[0025] Furthermore, in step S2, the polymerization reaction includes RAFT thermal polymerization or RAFT photopolymerization;

[0026] The reaction temperature for RAFT thermal polymerization is 60–90°C; the reaction temperature for RAFT photopolymerization is 20–30°C.

[0027] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The antimicrobial peptides prepared in this invention can effectively remove bacterial biofilms without inhibiting bacterial growth in both Gram-positive bacteria (such as Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative bacteria (such as Escherichia coli and Pseudomonas aeruginosa). They also exhibit excellent biocompatibility and are non-toxic. Attached Figure Description

[0029] Figure 1 This is a flowchart illustrating the preparation of antimicrobial peptides in Example 1;

[0030] Figure 2 N prepared in Example 1 β-AcryloylN α 1H NMR spectrum of -tert-butyloxycarbonyl-D-2,3-diaminopropionic acid monomer;

[0031] Figure 3 The proton NMR spectrum of the antimicrobial peptide prepared in Example 1;

[0032] Figure 4 The biofilm clearance effects of different types of antimicrobial peptides are shown in the images: (A) Optical photographs of biofilms; (B) Statistical results of biofilm clearance efficiency.

[0033] Figure 5 The diagram shows the effects of different types of antimicrobial peptides on biofilm components: (A) bacterial count; (B) biofilm protein composition; (C) biofilm polysaccharide composition.

[0034] Figure 6 To illustrate the biofilm clearance effects of antimicrobial peptides of different molecular weights, (A) optical photographs of biofilms; (B) statistical results of biofilm clearance efficiency. Detailed Implementation

[0035] This invention provides an antimicrobial peptide with the function of clearing bacterial biofilms, having the general structural formula shown in formula (I):

[0036]

[0037] Wherein, n is 5 to 100, preferably 10 to 80, and more preferably 20 to 60;

[0038] The structural formula of R is shown in equations (ii) to (v):

[0039]

[0040] In this invention, the antimicrobial peptide is used to remove bacterial biofilms, wherein the bacteria in the bacterial biofilm include Gram-negative bacteria and / or Gram-positive bacteria.

[0041] In this invention, Gram-negative bacteria are preferably Escherichia coli or Pseudomonas aeruginosa; Gram-positive bacteria include Staphylococcus aureus or Staphylococcus epidermidis.

[0042] In this invention, a mature biofilm model cultured on a titanium sheet is used for anti-biofilm detection. The specific model is as follows: a single bacterial colony is dynamically cultured in TSB until the late logarithmic growth stage. A sterile, dried titanium sheet is placed in a well plate, and sterile TSB diluted tenfold is added to the well plate. The plate is statically cultured at 37°C for 24 hours to obtain a mature biofilm. The culture medium is removed, and sterile TSB containing dissolved antimicrobial peptides is added. The control group uses only sterile TSB and is cultured for another 24 hours. The culture medium is removed, crystal violet is added for staining for 30 minutes, unfixed components are washed away, and 30% acetic acid is added to dissolve the unfixed components. The OD595 is measured to characterize the biofilm biomass.

[0043] The biofilm clearance rate is calculated using Equation (VI):

[0044]

[0045] This invention also provides a method for preparing the above-mentioned antimicrobial peptides, comprising the following steps:

[0046] S1. The amino acid, olefin compound and water are mixed and reacted to obtain the reaction product. The reaction product is then sequentially extracted, dried, rotary evaporated and vacuum dried to obtain olefin-functionalized amino acid monomer.

[0047] S2. An antimicrobial peptide is obtained by mixing olefin-functionalized amino acid monomers, chain transfer agents, catalysts and solvents and then carrying out a polymerization reaction.

[0048] In this invention, in step S1, the amino acid includes N α -tert-Butyloxycarbonyl-D-lysine, N α -tert-Butyloxycarbonyl-D-tyrosine, N ε -tert-Butyloxycarbonyl-D-lysine, N α -tert-Butyloxycarbonyl-D-2,3-diaminopropionic acid and N β Any one of -tert-butoxycarbonyl-D-2,3-diaminopropionic acid;

[0049] The olefin compound comprises acryloyl chloride and / or methacryloyl chloride, preferably acryloyl chloride.

[0050] In this invention, in step S1, the ratio of amino acids, olefin compounds and water is 800-1200 mg: 300-600 mg: 3-10 mL, preferably 900-1100 mg: 400-500 mg: 5-9 mL, and more preferably 950-1000 mg: 440-480 mg: 6-8 mL.

[0051] In this invention, in step S1, the reaction temperature is 0-30°C, preferably 5-25°C, and more preferably 10-20°C; the reaction time is 3-8 hours, preferably 4-7 hours, and more preferably 5-6 hours.

[0052] In this invention, the pH of the reaction system in step S1 is preferably 10.5.

[0053] In this invention, in step S2, the chain transfer agent includes one or more of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid, 2-(dodecylthiocarbonylthiothio)propionic acid, S-(2-cyano-2-propyl)-S-dodecyltrithiocarbonyl ester, and 2,2'-[methylthiobis(thio)]bis[2-methylpropionic acid];

[0054] The catalyst includes one or more of 2′,4′,5′,7′-tetrabromofluorescein, tetraphenylporphyrin zinc, tris(2-phenylpyridine)iridium, sodium bicarbonate, ammonium persulfate, and azobisisobutyronitrile;

[0055] The solvent includes one or more of ethyl acetate, methanol, ethanol, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, concentrated hydrochloric acid, and hydrogen peroxide.

[0056] In this invention, in step S2, the molar ratio of olefin-functionalized amino acid monomer to chain transfer agent is 20-150:1, preferably 40-120:1, and more preferably 60-100:1;

[0057] The molar ratio of the olefin-functionalized amino acid monomer to the catalyst is 10. 3 ~10 5 :1, preferably 10 4 :1;

[0058] The mass-to-volume ratio of the olefin-functionalized amino acid monomer to the solvent is 0.5–1.5 g: 7 mL, preferably 0.8–1.2 g: 7 mL, and more preferably 1.0 g: 7 mL.

[0059] In this invention, the polymerization reaction time in step S2 is 4 to 12 hours, preferably 5 to 10 hours, and more preferably 6 to 8 hours.

[0060] In this invention, in step S2, deoxygenation is performed before the polymerization reaction, and the deoxygenation time is 40-60 min, preferably 50 min.

[0061] In this invention, the polymerization reaction in step S2 includes RAFT thermal polymerization or RAFT photopolymerization;

[0062] The reaction temperature for RAFT thermal polymerization is 60–90°C, preferably 65–85°C, and more preferably 70–80°C; the reaction temperature for RAFT photopolymerization is 20–30°C, preferably 22–28°C, and more preferably 24–26°C.

[0063] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1

[0065] The flowchart for preparing the antimicrobial peptide in this embodiment is as follows: Figure 1 As shown.

[0066] Weigh 1000mg N α -tert-Butoxycarbonyl-D-2,3-diaminopropionic acid was dissolved in 5 mL of deionized water, and the pH was adjusted to 10.5 with sodium hydroxide solution. The solution was stirred and dissolved in an ice bath. Then, 532 mg of acryloyl chloride was slowly added dropwise to the system while maintaining the pH at 10.5. After 30 min, the ice bath was removed, and the reaction was carried out at 25 °C for 4 h. Subsequently, the pH was adjusted to 2.9, and the mixture was extracted three times with ethyl acetate and dried over anhydrous sodium sulfate for 8 h. Finally, the solvent was removed by rotary evaporation, and the mixture was dried under vacuum for 24 h to obtain N. β -Acryloyl-N α -tert-Butoxycarbonyl-D-2,3-diaminopropionic acid monomer.

[0067] 10g N β -Acryloyl-N α The tert-butyloxycarbonyl-D-2,3-diaminopropionic acid monomer was added to a quartz bottle and dissolved in 70 mL of dimethyl sulfoxide. The monomer-to-chain transfer agent molar ratio was 60:1, and the monomer-to-catalyst molar ratio was 10:1. 5 1. Add 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid and azobisisobutyronitrile to a quartz bottle and seal the quartz bottle with a rubber stopper. Then, purge the bottle with nitrogen gas for 50 min using long and short needles and seal the needle opening. Perform RAFT thermal polymerization at 60℃ for 10 h to obtain an antimicrobial peptide (1).

[0068] The prepared N was analyzed using a nuclear magnetic resonance spectrometer (Bruker AVANCE 300MHz NMR spectrometer, Bruker, Switzerland). β -Acryloyl-N α -tert-Butoxycarbonyl-D-2,3-diaminopropionic acid monomer ( Figure 2 ) and antimicrobial peptides ( Figure 3 Characterized by ) Figure 2 and Figure 3 The monomer and polymer were successfully prepared, and the degree of polymerization of the polymer was 33.

[0069] Example 2

[0070] Weigh out 800mg N β-tert-Butoxycarbonyl-D-2,3-diaminopropionic acid was dissolved in 5 mL of deionized water, and the pH was adjusted to 10.5 with sodium hydroxide solution. The solution was stirred and dissolved in an ice bath. Then, 532 mg of acryloyl chloride was slowly added dropwise to the system while maintaining the pH at 10.5. After 30 min, the ice bath was removed, and the reaction was carried out at 25 °C for 4 h. Subsequently, the pH was adjusted to 2.9, and the mixture was extracted three times with ethyl acetate and dried over anhydrous sodium sulfate for 8 h. Finally, the solvent was removed by rotary evaporation, and the mixture was dried under vacuum for 24 h to obtain N. α -Acryloyl-N β -tert-Butoxycarbonyl-D-2,3-diaminopropionic acid monomer.

[0071] 12g N α -Acryloyl-N β The tert-butyloxycarbonyl-D-2,3-diaminopropionic acid monomer was added to a quartz bottle and dissolved in 70 mL of dimethyl sulfoxide. The monomer-to-chain transfer agent molar ratio was 60:1, and the monomer-to-catalyst molar ratio was 10:1. 5 1. Add 2-(dodecylthiocarbonylthiothiothio)propionic acid and sodium bicarbonate to a quartz bottle and seal the quartz bottle with a rubber stopper. Then, purge the bottle with nitrogen gas for 50 min using long and short needles and seal the needle opening. Perform RAFT thermal polymerization at 70℃ for 8 h to obtain an antimicrobial peptide (2).

[0072] Example 3

[0073] Weigh out 1200mg N α -tert-Butyloxycarbonyl-D-lysine was added to 5 mL of deionized water, and the pH was adjusted to 10.5 with sodium hydroxide solution. The mixture was dissolved under stirring in an ice bath. Then, 393.2 mg of acryloyl chloride was slowly added dropwise to the system while maintaining the pH at 10.5. After 30 min, the ice bath was removed, and the reaction was carried out at 25 °C for 4 h. Subsequently, the pH was adjusted to 2.9, and the mixture was extracted three times with ethyl acetate and dried over anhydrous sodium sulfate for 8 h. Finally, the solvent was removed by rotary evaporation, and the mixture was dried under vacuum for 24 h to obtain N. ε -Acryloyl-N α -tert-Butoxycarbonyl-D-lysine monomer.

[0074] 9g N ε -Acryloyl-N α The tert-butyloxycarbonyl-D-lysine monomer was added to a quartz bottle and dissolved in 70 mL of dimethyl sulfoxide. The monomer-to-chain transfer agent molar ratio was 60:1, and the monomer-to-catalyst molar ratio was 10:1. 5 1. Add 2-(dodecylthiocarbonylthiothiothio)propionic acid and zinc tetraphenylporphyrin to a quartz bottle and seal the quartz bottle with a rubber stopper. Then, purge the bottle with nitrogen gas for 50 min using long and short needles and seal the needle opening. Perform RAFT thermal polymerization at 80℃ for 6 h to obtain an antimicrobial peptide (3).

[0075] Example 4

[0076] Weigh 1000mg N ε -tert-Butyloxycarbonyl-D-lysine was added to 5 mL of deionized water, and the pH was adjusted to 10.5 with sodium hydroxide solution. The mixture was dissolved under stirring in an ice bath. Then, 393.2 mg of acryloyl chloride was slowly added dropwise to the system while maintaining the pH at 10.5. After 30 min, the ice bath was removed, and the reaction was carried out at 25 °C for 4 h. Subsequently, the pH was adjusted to 2.9, and the mixture was extracted three times with ethyl acetate and dried over anhydrous sodium sulfate for 8 h. Finally, the solvent was removed by rotary evaporation, and the mixture was dried under vacuum for 24 h to obtain N. α -Acryloyl-N ε -tert-Butoxycarbonyl-D-lysine monomer.

[0077] 15g N α -Acryloyl-N ε The tert-butyloxycarbonyl-D-lysine monomer was added to a quartz bottle and dissolved in 70 mL of dimethyl sulfoxide. The monomer-to-chain transfer agent molar ratio was 60:1, and the monomer-to-catalyst molar ratio was 10:1. 4 1. Add 2-(dodecylthiocarbonylthiothiothio)propionic acid and zinc tetraphenylporphyrin to a quartz bottle and seal the quartz bottle with a rubber stopper. Then, purge the bottle with nitrogen gas for 50 min using long and short needles and seal the needle openings. Perform RAFT thermal polymerization at 80℃ for 6 h to obtain an antimicrobial peptide (4).

[0078] Example 5

[0079] Weigh 1000mg N α -tert-Butyloxycarbonyl-D-tyrosine was added to 5 mL of deionized water, and the pH was adjusted to 10.5 with sodium hydroxide solution. The mixture was dissolved under stirring in an ice bath. Then, 593 mg of methacryloyl chloride was slowly added dropwise to the system while maintaining the pH at 10.5. After 30 min, the ice bath was removed, and the reaction was carried out at 25 °C for 4 h. Subsequently, the pH was adjusted to 3.8, and the mixture was extracted three times with ethyl acetate and dried over anhydrous sodium sulfate for 8 h. Finally, the solvent was removed by rotary evaporation, and the mixture was dried under vacuum for 24 h to obtain O-methacryloyl-N α -tert-Butoxycarbonyl-D-tyrosine monomer.

[0080] 8g of O-methacryloyl-N α The tert-butyloxycarbonyl-D-tyrosine monomer was added to a quartz bottle and dissolved in 70 mL of dimethyl sulfoxide. The monomer-to-chain transfer agent molar ratio was 60:1, and the monomer-to-catalyst molar ratio was 10:1. 31. S-(2-cyano-2-propyl)-S-dodecyl trithiocarbonyl ester and sodium bicarbonate were added to a quartz bottle, which was then sealed with a rubber stopper. Nitrogen gas was then purged through long and short needles for 50 minutes to remove oxygen, and the needle openings were sealed. RAFT thermal polymerization was carried out at 70°C for 9 hours to obtain an antimicrobial peptide.

[0081] Example 6

[0082] Similar to Example 1, except that the molar ratio of monomer to chain transfer agent is 20:1, and an antimicrobial peptide (5) is prepared.

[0083] Example 7

[0084] Similar to Example 1, except that the molar ratio of monomer to chain transfer agent is 40:1, and an antimicrobial peptide (6) is prepared.

[0085] Example 8

[0086] Similar to Example 1, except that the molar ratio of monomer to chain transfer agent is 80:1, and an antimicrobial peptide (7) is prepared.

[0087] Example 9

[0088] Similar to Example 1, except that the molar ratio of monomer to chain transfer agent was 120:1, and an antimicrobial peptide (8) was prepared.

[0089] Example 10

[0090] Similar to Example 1, except that the molar ratio of monomer to chain transfer agent is 150:1, and an antimicrobial peptide (9) is prepared.

[0091] Application Example 1

[0092] Construction of a mature biofilm clearance model: A single colony of MRSA1857 was dynamically cultured in TSB until the late logarithmic growth phase. A sterile, dried titanium sheet was placed in a well plate, diluted 10-fold with sterile TSB, and added to the well plate. The plate was statically cultured at 37°C for 24 h to obtain a mature biofilm. The culture medium was removed, and sterile TSB containing a soluble antimicrobial peptide (512 μg / mL) was added. The control group used only sterile TSB and was cultured for another 24 h. The culture medium was removed, and crystal violet was added for staining for 30 min. The unfixed components were washed away, dissolved in 30% acetic acid, and OD595 was measured to characterize the biofilm biomass.

[0093] The biofilm clearance rate is calculated using Equation (VI):

[0094]

[0095] The biofilm clearance rate of the antimicrobial peptides was calculated using equation (VI), and the specific data are as follows: Figure 4As shown, the biofilm clearance efficiencies of antimicrobial peptides 1-4 were 57.7%, 47.5%, 37.0%, and 39.2%, respectively. Furthermore, Figure 5 The results showed that antimicrobial peptides 1 and 3 mainly achieve biofilm disintegration by scavenging polysaccharides in biofilms, with little impact on the protein components of biofilms.

[0096] Application Example 2

[0097] Construction of a mature biofilm clearance model: A single colony of S. aureus was dynamically cultured in TSB until the late logarithmic growth phase. A sterile, dried titanium sheet was placed in a well plate, diluted 10-fold with sterile TSB, and added to the well plate. The plate was statically cultured at 37°C for 24 h to obtain a mature biofilm. The culture medium was removed, and sterile TSB containing a soluble antimicrobial peptide (512 μg / mL) was added. The control group used only sterile TSB and was cultured for another 24 h. The culture medium was removed, crystal violet was added for staining for 30 min, unfixed components were washed away, and the biofilm was dissolved in 30% acetic acid. The OD595 was measured to characterize the biofilm biomass.

[0098] The biofilm clearance rate is calculated using Equation (VI):

[0099]

[0100] The biofilm clearance rate of the antimicrobial peptides was calculated using equation (VI), and the specific data are as follows: Figure 6 As shown, the biofilm clearance efficiencies of antimicrobial peptides 5, 6, 1, 7, 8, and 9 were 68.6%, 65.4%, 68.8%, 62.6%, 61.2%, and 63.3%, respectively.

[0101] Application Example 3

[0102] Construction of a mature biofilm clearance model: A single colony of P. aeruginosa was dynamically cultured in TSB until the late logarithmic growth phase. A sterile, dried titanium sheet was placed in a well plate, and sterile TSB diluted 10-fold was added to the well plate. The plate was statically cultured at 37°C for 24 h to obtain a mature biofilm. The culture medium was removed, and sterile TSB containing a soluble antimicrobial peptide (512 μg / mL) was added. The control group used only sterile TSB and was cultured for another 24 h. The culture medium was removed, and crystal violet was added for staining for 30 min. The unfixed components were washed away, and 30% acetic acid was added to dissolve the unfixed components. The OD595 was measured to characterize the biofilm biomass.

[0103] The biofilm clearance rate is calculated using Equation (VI):

[0104]

[0105] The biofilm clearance rates of antimicrobial peptides were calculated using Equation (VI). The biofilm clearance efficiencies of antimicrobial peptides 5 and 6 were 41.4% and 40.9%, respectively.

[0106] Application Example 4

[0107] Construction of a mature biofilm clearance model: E. coli single colonies were dynamically cultured in TSB until the late logarithmic growth phase. Sterile dried titanium sheets were placed in well plates, and sterile TSB diluted 10-fold was added to the well plates. The plates were statically cultured at 37°C for 24 h to obtain a mature biofilm. The culture medium was removed, and sterile TSB containing a soluble antimicrobial peptide (512 μg / mL) was added. The control group used only sterile TSB and was cultured for another 24 h. The culture medium was removed, crystal violet was added for staining for 30 min, unfixed components were washed away, and 30% acetic acid was added to dissolve the unfixed components. OD595 was measured to characterize biofilm biomass.

[0108] The biofilm clearance rate is calculated using Equation (VI):

[0109]

[0110] The biofilm clearance rate of the antimicrobial peptide was calculated using Equation (VI), and the biofilm clearance efficiency of the antimicrobial peptide 7 was 45.5%.

[0111] Application Example 5

[0112] Construction of a mature biofilm clearance model: A single colony of *S. epidermidis* was dynamically cultured in TSB until the late logarithmic growth phase. A sterile, dried titanium sheet was placed in a well plate, diluted 10-fold with sterile TSB, and added to the well plate. The plate was statically cultured at 37°C for 24 h to obtain a mature biofilm. The culture medium was removed, and sterile TSB containing a soluble antimicrobial peptide (512 μg / mL) was added. The control group used only sterile TSB and was cultured for another 24 h. The culture medium was removed, and crystal violet was added for staining for 30 min. The unfixed components were washed away, dissolved in 30% acetic acid, and OD595 was measured to characterize the biofilm biomass.

[0113] The biofilm clearance rate is calculated using Equation (VI):

[0114]

[0115] The biofilm clearance rate of the antimicrobial peptide was calculated using Equation (VI), and the biofilm clearance efficiency of antimicrobial peptide 1 was 63.0%.

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An anti-bacterial peptide-like substance having a function of removing a bacterial biofilm, characterized by comprising a peptide having an amino acid sequence represented by SEQ ID NO:

1. Has the structural general formula as shown in formula (I): Formula (I); Wherein, n is 5~100; R structural formula as shown in formula (II)~(V): Formula (II), formula (III), formula (IV), formula (V); The class antibacterial peptide has the effect of removing biofilm without inhibiting bacterial growth.

2. Use of the antiseptic peptide-like peptide according to claim 1 for eradicating bacterial biofilm, characterized in that, The bacteria in the bacterial biofilm include gram-negative bacteria and / or gram-positive bacteria.

3. Use of the antifungal peptide-like peptide according to claim 2 for eradicating bacterial biofilm, characterized in that, The preparation method of the class antibacterial peptide comprises the following steps: S1, the amino acid, olefin compound and water are mixed and reacted to obtain a reaction product, and the reaction product is sequentially extracted, dried, rotary evaporated and vacuum dried to obtain an olefin functionalized amino acid monomer; S2, the olefin functionalized amino acid monomer, chain transfer agent, catalyst and solvent are mixed and polymerized to obtain the class antibacterial peptide.

4. Use of the peptide as claimed in claim 3 for eradicating bacterial biofilm, characterized in that, The amino acid in step S1 includes N α - tert-butyloxycarbonyl-D-lysine, N ε - tert-butyloxycarbonyl-D-lysine, N α - tert-butyloxycarbonyl-D-tyrosine, N α - tert-butyloxycarbonyl-D-2,3-diaminopropionic acid, and N β - tert-butyloxycarbonyl-D-2,3-diaminopropionic acid; The olefin compound comprises acryloyl chloride.

5. Use of the antifungal peptide-like peptide according to claim 4 for eradicating bacterial biofilm, characterized in that, In the step S1, the amount ratio of the amino acid, the olefin compound and the water is 800~1200mg:300~600mg:3~10mL.

6. Use of an antiseptic peptide-like peptide according to any one of claims 3 to 5 for the removal of bacterial biofilms, characterized in that, In the step S1, the reaction temperature is 0~30℃, and the reaction time is 3~8h.

7. Use of the antiseptic peptide-like peptide according to claim 6 for eradicating bacterial biofilm, characterized in that, In the step S2, the chain transfer agent includes one or more of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid, 2-(dodecylthiocarbonylthiothiothio)-propionic acid, S-(2-cyano-2-propyl)-S-dodecyltrithiocarbonyl ester and 2,2'-[methylthio bis(thio)] bis[2-methylpropionic acid]; The catalyst includes one or more of 2',4',5',7'-tetrabromofluorescein, zinc tetraphenylporphyrin, tris(2-phenylpyridine)iridium, sodium bicarbonate, ammonium persulfate and azobisisobutyronitrile; The solvent includes one or more of ethyl acetate, methanol, ethanol, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, concentrated hydrochloric acid and hydrogen peroxide.

8. Use of the antiseptic peptide-like peptide according to claim 4, 5 or 7 for eradicating bacterial biofilm, characterized in that, In the step S2, the molar ratio of the olefin functionalized amino acid monomer and the chain transfer agent is 20~150:1; The molar ratio of the olefin-functionalized amino acid monomer and the catalyst is 10 3 ~10 5 : 1; The mass-volume ratio of the olefin functionalized amino acid monomer and the solvent is 0.5~1.5g:7mL.

9. Use of the antiseptic peptide-like peptide according to claim 8 for eradicating bacterial biofilm, characterized in that, In the step S2, the polymerization reaction time is 4~12h.

10. Use of the antiseptic peptide-like peptide according to claim 9 for eradicating bacterial biofilm, characterized in that, In the step S2, the polymerization reaction includes RAFT thermal polymerization or RAFT photopolymerization; The reaction temperature of the RAFT thermal polymerization is 60~90℃, and the reaction temperature of the RAFT photopolymerization is 20~30℃.

Images